LOCUS Exported 7042 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS TALEN-ID12-T SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7042) AUTHORS Schmid-Burgk JL, Schmidt T, Kaiser V, Honing K, Hornung V TITLE A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. JOURNAL Nat Biotechnol. 2012 Dec 16. doi: 10.1038/nbt.2460. PUBMED 23242165 REFERENCE 2 (bases 1 to 7042) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7042 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 913..931 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 980..1045 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1052..1072 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1831..2418 /codon_start=1 /product="nonspecific DNA cleavage domain of the FokI endonuclease (Li et al., 1992)" /label=FokI cleavage domain /note="must dimerize to be catalytically active" /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" protein_bind 2440..2473 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter complement(2594..2612) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2595..2612) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2632..2649) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2638..2862 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2908..3336 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2995..3014) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3205..3226 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3345..3365) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3350..3680 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3531..3666 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3593..3612 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3747..4541 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3801..3820) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4411..4430 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4715..4836 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4752..4771) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4806..4825 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4885..4901) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4885..4901) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4898..4920) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4909..4925 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4933..4963) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4978..4999 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5116..5133) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5287..5875) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5367..5386) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6046..6906) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6669..6688 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6907..7011) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggaccc 901 tcgtacagaa gctaatacga ctcactatag ggaaataaga gagaaaagaa gagtaagaag 961 aaataataga ctcgagatgg actacaagga ccacgacggc gactacaagg accacgacat 1021 cgactacaag gacgacgacg acaagatggc ccccaagaag aagagaaagg tgggcatcca 1081 cagaggcgtg cccatggtgg acctgagaac cctgggctac agccagcagc agcaggagaa 1141 gatcaagccc aaggtgagaa gcaccgtggc ccagcaccac gaggccctgg tgggccacgg 1201 cttcacccac gcccacatcg tggccctgag ccagcacccc gccgccctgg gcaccgtggc 1261 cgtgaagtac caggacatga tcgccgccct gcccgaggcc acccacgagg ccatcgtggg 1321 cgtgggcaag cagtggagcg gcgccagagc cctggaggcc ctgctgaccg tggccggcga 1381 gctgagaggc ccccccctcc agctggacac cggccagctg ctgaagatcg ccaagagagg 1441 cggcgtgacc gccgtggagg ccgtgcacgc ctggagaaac gccctgaccg gcgcccccct 1501 gaatttgaca cccgaccaag gtaccggtgg atccggtggt accggttgct ccctgtcttg 1561 tgtcaagacc atgggctgac ccccgagcag gtggtggcca tcgccagcaa cggcggcggc 1621 agacccgccc tggagagcat cgtggcccag ctgagcagac ccgaccccgc cctggccgcc 1681 ctgaccaacg accacctggt ggccctggcc tgcctgggcg gcagacccgc cctggacgcc 1741 gtgaagaagg gcctgcccca cgcccccgcc ctgatcaaga gaaccaacag aagaatcccc 1801 gagagaacca gccacagagt ggccggcagc cagctggtga agagcgagct ggaggagaag 1861 aagagcgagc tgagacacaa gctgaagtac gtgccccacg agtacatcga gctgatcgag 1921 atcgccagaa acagcaccca ggacagaatc ctggagatga aggtgatgga gttcttcatg 1981 aaggtgtacg gctacagagg caagcacctg ggcggcagca gaaagcccga cggcgccatc 2041 tacaccgtgg gcagccccat cgactacggc gtgatcgtgg acaccaaggc ctacagcggc 2101 ggctacaacc tgcccatcgg ccaggccgac gagatgcaga gatacgtgga ggagaaccag 2161 accagaaaca agcacatcaa ccccaacgag tggtggaagg tgtaccccag cagcgtgacc 2221 gagttcaagt tcctgttcgt gagcggccac ttcaagggca actacaaggc ccagctgacc 2281 agactgaacc acatcaccaa ctgcaacggc gccgtgctga gcgtggagga gctgctgatc 2341 ggcggcgaga tgatcaaggc cggcaccctg accctggagg aggtgagaag aaagttcaac 2401 aacggcgaga tcaacttcag aagctctaga tgaactagta taacttcgta tagcatacat 2461 tatacgaagt tatgctagcg cggccgcgct gccttctgcg gggcttgcct tctggccatg 2521 cccttcttct ctcccttgac acctgtacct cttggtcttt gaataaagcc tgagtaggaa 2581 gtgagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg 2641 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 2701 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 2761 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 2821 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 2881 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 2941 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 3001 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3061 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3121 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 3181 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 3241 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa 3301 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 3361 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca 3421 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 3481 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 3541 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 3601 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 3661 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 3721 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 3781 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 3841 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 3901 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3961 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 4021 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 4081 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 4141 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 4201 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 4261 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 4321 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 4381 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 4441 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 4501 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 4561 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 4621 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 4681 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 4741 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 4801 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 4861 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 4921 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4981 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 5041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 5101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 5161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 5221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5461 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5701 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 7021 ccgaaaagtg ccacctgacg tc //